To date, efforts have been undertaken to develop culture conditions to maximize cell culture growth and thereby increase resultant product yield. Early work in the development of animal cell culture media focused on the formulation of such media to achieve rapid cell proliferation (White, P. R., 1946, Growth, 10:231-289, and Waymouth, C., 1974, J. Natl. Cancer Inst., 53:1443-1448). Such media incorporate specific nutrients especially sugars, amino acids, vitamins, salts, and in some cases trace metal ions, purines, and pyrimidines. These media are most often supplemented with animal serum. Today some of the more widely used basal media for mammalian cell cultures include Hams F-12, Dulbecco's modified Eagle's medium (DME), RPMI 1640, and Iscove's modified DME.
Production of human monoclonal antibodies using hybridoma cell lines will be used in this application as an example for product expression in cell culture.
Culture media have been previously described which were developed specifically for low serum and serum-free mammalian cell cultures for production of monoclonal antibodies. One such serum-free medium is disclosed in European Patent Publication 076,647, published Apr. 13, 1983. Other media have been developed by changing levels of supplements such as trace elements, and vitamins and incorporating purified protein hormone additives. References to such media include, for example, Barnes, D., et al., 1980 Cell, 22:649-655; Cleveland, W. L., et al., 1983, J. Immunol. Meth., 56:221-234; Iscove, N., et al., 1978, J. Exp. Med., 147:923-933; Kawamoto, T., et al., 1983, Analytical Biochemistry, 130:445-453; Kovar, J., et al., 1986, Immunology Letters, 7:339-345; Murakami, H., et al., 1983, Argic. Biol. Chem., 47(8):1835-1840; Murakami, H., et al., 1982, Proc. Natl. Acad. Sci. USA, 79:1158-1162; Muzik, H., et al., 1982, In Vitro, 18:515-524; and Wolpe, S. D., "In Vitro Immunization and Growth of Hybridomas in Serum-Free Medium", in J. P. Mather, ed., Mammalian Cell Culture, Plenum Press, New York, 1984; Hagiwara, H., et al., 1985, 117-122 in H. Murakami et al. (eds) Growth and Differentiation of Cells in Defined Environment, Springer-Verlag, Berlin, 1985; Tharakan, J. P., et al., 1986, J. Immunol. Meth., 94:225-235; Cole, S.P.C., 1987, J. Immunol. Meth., 97:29-35; McHugh, Y. E., 1983, BioTechniques, June/July issue:72-77. Components which are common to most if not all these media include glucose at concentrations up to 4.5 g/L, glutamine at concentrations of 2-4 mM, choline generally at about 1-4 mg/L, tryptophan and other amino acids. Tryptophan is generally present at concentrations less than 20 mg/L. Several of these media also contain the growth factors insulin and transferrin.
Efforts to increase antibody yield have focused primarily on means to optimize cell growth and cell density. As a general point of reference, antibody titres from murine hybridoma cell lines are highly variable from cell line to cell line and range typically from 10 to 350 mg/L (Lambert, K. J., et al., 1987, Dev. Indust. Microbiol, 27:101-106). Human monoclonal antibody expression from human/human or human/mouse fusions are also highly variable from cell line to cell line and range typically from 0.1 to 25 mg/L (Hubbard, R., Topics in Enzyme and Fermentation Biotechnology, Chapt. 7:196-263, Wisemand, A., ed., John Wiley & Sons, New York (1983). These values are indicative of culture conditions that are optimized for cell growth.
Another approach from the literature to increasing product production is to achieve high cell densities by cell recycle or entrapment methods. Examples of these methods include hollow fiber reactors (Altshuler, G. L., et al., 1986, Biotech. Bioeng., XXVIII:646-658; ceramic matrix reactors (Marcipar, A., et al., 1983, Annals. N.Y. Acad. Sci., 413:416-420; Nature, 302:629-630); perfusion reactors (Feder, J., et al., 1985, American Biotech. Laboratory, III:24-36) and others.
While a variety of methods to increase product expression from cell culture are being explored, the primary focus is still on the optimization of cell growth. In typical culture media the culture dies rapidly after maximum cell density is reached.
Another example form the literature documents that, at least for some cell lines, product (monoclonal antibody) production proceeds even after a culture stops growing (Velez, D., et al., 1986, J. Immunol. Meth., 86:45-52; Reuveny, S., et al., 1986, ibid at 53-59). Arathoon, W., et al., 1986, Science, 232:1390-1395 reported that a 1,000 liter hybridoma fermentation produced about 80 grams of monoclonal antibody during the growth phase and another 170 grams of antibody during an extended stationary/death phase. J. Birth, et al., (European Patent Application No. 87/00195, 1987) describe a procedure of Fed-batch culture wherein nutrients are added to a culture over time and culture longevity is increased. Final antibody titres from the culture are thus increased.
Thus, it will be appreciated that there is a critical need for media that will support the growth of animal cells and stimulate the production of products, including antibodies, and other natural or recombinant protein products to greater levels than can be realized using media that are currently available.